Iron-sulfur (Fe-S) proteins are a group of functionally diverse proteins that contain prosthetic groups composed of Fe and sulfur of various structures, termed Fe-S clusters. They have a well established functional role of mediating biological electron transfer in the respiratory and photosynthetic electron transfer chains and are involved in the metabolism of essential organic elements. They are also involved in a diverse range of non-redox processes including sensing and regulatory function. This proposal seeks support to continue the PI's research project of employing a combined Mossbauer and EPR spectroscopic approach together with the rapid freeze-quench technique to investigate (1) the biosynthesis of Fe-S clusters and (2) the newly emerged functions of Fe-S clusters found in two classes of Fe-S enzymes: ferredoxin-dependent disulfide reductases and S-adenosylmethionine (SAM)-dependent Fe-S enzymes. At present, there are three known Fe-S cluster biosynthesis machineries: the nitrogen fixation specific NIF system, the ubiquitous "housekeeping" iron-sulfur cluster assembly ISC system, and the newly discovered "sulfur mobilization" SUF system. This research project focuses on the NIF and ISC systems. Experiments are designed to investigate the mechanism that the NIF and ISC systems use for cluster assembly and transport. The emphasis is on the transport of the assembled clusters from the scaffold proteins to the targeted proteins. In addition, the in vivo functional roles of the six isc gene products will be investigated by using whole cell Mossbauer spectroscopy and a controlled bacterial expression system that permits real-time depletion of each of the six proteins. For the studies of the novel functions of Fe-S clusters, three functionally diverse enzymes were chosen initially. They are, ferredoxin:thioredoxin reductase (FTR), pyruvate formate-lyase-activating enzyme (PFL-AE), and biotin synthase (BioB). FTR catalyzes the reductive cleavage of disulfide groups in thioredoxins for enzyme activation. PFL-AE activates pyruvate formate lyase (PFL) by catalyzing the generation of a glycyl radical in PFL, and BioB converts dethiobiotin to biotin. Significant progress has been made during the current budget period in understanding the functions of the Fe-S clusters in these enzymes. The results have established that all three enzymes employ a unique site-specific Fe-based Fe4S4 cluster chemistry for their respective functions. In an effort to further determine the detailed mechanistic steps involved in the catalytic cycles of these enzymes, rapid freeze-quench and cryoreduction techniques will be used to trap reaction intermediates for spectroscopic characterization and kinetic investigations. In addition, we propose to extend our study to include another important SAM-dependent enzyme, the human MOCS1A, which catalyzes the initial steps in the biosynthesis of molybdenum cofactor, MoCo. It is by studying these functionally diverse enzymes that we hope to identify factors that are essential for controlling the reactivity of Fe-S clusters.